Separatory material with the use of stimulus-responsive polymer and separation method by using the separatory material

ABSTRACT

A novel method for separating a target substance, for example, metal ion, drug or biological component is provided. According to the method, the surface of a packing undergoes a chemical or physical environmental change under a physical stimulus so that the interaction of a substance interacting with the target substance is reversibly changed in an aqueous solution, thus effecting separation.

TECHNICAL FIELD

This invention relates to a novel separation method for separating atarget substance (metal ions, drugs, biological components, etc.) withthe use of a separatory material wherein the interaction force of asubstance (ligand) interacting with the target substance can bereversibly changed in an aqueous system due to a structural change or apolarity change of a stimulus-responsive polymer under a physicalstimulus, and a separatory material such as a packing to be used in thismethod.

BACKGROUND OF INVENTION

The most effective and efficient means employed at present forseparating and purifying biological components and drugs include ionexchange chromatography, reversed phase chromatography, affinitychromatography, etc. In recent years, bioengineering procedures havemade remarkable advances and physiologically active substances(recombinant proteins, glycoproteins, etc.) are produced thereby on amass scale. Under these circumstances, there is a growing requirementfor methods by which these products can be quickly and efficientlyseparated and purified without inactivation.

In the chromatographic techniques as cited above, however, targetsubstances (biological components, drugs, recombinant proteins,glycoproteins, etc.) are eluted by changing the salt concentration,organic solvent concentration, pH value, etc. of eluents. It is knownthat, in such a case, the pH value, organic solvent, etc. frequentlybring about severe conditions for the target substance and thus lowerthe recovery yield thereof. In addition, the salt, organic solvent,pH-regulating agent, etc. employed in eluting the target substanceshould be finally eliminated by desalting, drying, etc. Therefore, anadditional step is required, after the completion of the separation andpurification of the target substance, to perform an operation foreliminating the salt, organic solvent, pH-regulating agent, etc. As aresult, the activity and yield of the final product are often lowered.

When the target substance is eluted by a chemical means with the use ofa salt, an organic solvent, a pH-regulating agent, etc., the chemical(i.e., the salt, organic solvent, pH-regulating agent, etc.) containedin the eluent causes the above-mentioned problems of inactivation, adecrease in the yield, etc. It is expected that if a physical changeinduced by, for example, heat, light or a magnet could affect theelution of a target substance, the target substance would be eluted notby a chemical means but by a physical one, thus solving the problems ofinactivation, a decrease in the yield, etc.

Recently, separation material comprising stimulus-responsive polymerscovalently attached to ion exchanging groups have been described. Seefor instance JP application 140722/98 and corresponding WO applicationWO99/61904.

Galaev et al (J. Chromatog. A 684 (1994) 37-43 describe temperatureelution of lactate dehydrogenase (LDH) in a chromatographic system inwhich a dye (ligand) is covalently attached to a base matrix which alsocarries a physically adsorbed temperature responsive polymer.

Hofman et al., (WO 8706152) describes a separation method in which theligand is attached to a temperature responsive polymer. Binding andelution of the target substance occur at the same side of the criticalsolution temperature.

There are also a number of publications describing chromatography basedon separatory material comprising stimulus-responsive polymers butwithout having a ligand covalently attached to thetemperature-responsive polymer. Gewehr et al (Macromolecular Chemistryand Physics 193 (1992) 249-256) describes gel chromatography on poroussilica beads coated with a temperature-responsive polymer. Hosoya et al(Anal. Chem. 67 (1995) 1907-1911); Yamamoto et al. (Proc. 114^(th)National Meeting of the Pharmaceutical Society of Japan, Tokyo (1994)160; Kanazawa et al (Yakugaku Zasshi 117 (10-11) (1997) 817-824;Kanazawa et al (Anal. Chem. 68(1) (1996) 100-105); Kanazawa et al (Anal.Chem. 69(5) (1997) 823-830); Kanazawa et al (J. Pharm. Biomed. Anal. 15(1997) 1545-1550); Yakushiji et al (Langmuir 14(16) 1998) 4657-466268);Kanazawa et al (Trends Anal. Biochem. 17(7) (1998) 435-440); Yakushijiet al (Anal. Chem. 71(6) 1999) 1125-1130); Grace & Co (EP 534016); Okano(JP 6-108643) describe reversed phase chromatography on matrices coveredby a thermoresponsive olymer for the separation of biomolecules. Thematrices may be porous. The hydrophobic groups utilized are inherent inthe polymer as such. There is no ligand that has been covalentlyattached to the polymer after polymerisation.

SUMMARY OF INVENTION

From this point of view, the present inventors have conducted intensivestudies and developments on the elution of a target substance by aphysical means to thereby solve the above problems. As a result, theysynthesized a composite material comprising a stimulus-responsivepolymer with a ligand molecule by binding poly(N-isopropylacrylamide) toa molecule (i.e., a ligand molecule) capable of interacting with atarget substance. Subsequently they have found out that use of thiscomposite material makes it possible to obtain a separatory materialwhich undergoes a change in the interaction between the ligand moleculeand the target substance under a physical stimulus. The presentinvention has been completed based on this finding.

The present invention relates to a method for separating substancescharacterized in that a composite material comprising astimulus-responsive polymer and a substance (ligand) interacting with atarget substance undergoes a physical or chemical change of thestimulus-responsive polymer under a physical stimulus so that theenvironment of the interaction between the target substance and themolecule interacting therewith is physically or chemically changed. Thismeans that the target substance can be released from the ligand and alsofrom the separatory material, thus effecting separation of the targetsubstance from the composite material or separatory material.

The present invention further relates to a method for separatingsubstances characterized by comprising (a) binding a target substance ona stationary phase of a separatory material (including chromatographicpacking) chemically modified with a composite material comprising astimulus-responsive polymer and a substance (ligand) interactingspecifically with the target substance; then (b) changing continuouslyor stepwise the temperature, preferably by external means, to therebyweaken the interaction between the ligand and the target substance; andeluting the chromatographic packing by a mobile phase while maintaininga temperature which permit a weakened interaction between the ligand andthe target substance thus effecting separation. The mobile phase may bea liquid, for instance aqueous.

The present invention further relates to a separatory material (forexample, a chromatographic packing) wherein the ability of a substanceto recognize a molecule can be changed due to a physical stimulus.

Another embodiment of the invention is a method for the separation ofone or more target substances from a liquid. This embodiment comprisesthe steps of substances from

(a) bringing a liquid sample (I) containing a target substance incontact with a separation medium/separatory material (including achromatographic packing) which is functionalized with a ligand which iscapable of binding to the target substance, said contact being underconditions permitting binding of said target substance to said ligand;

(b) contacting said carrier with a liquid (II) not containing said atleast one target substance under conditions such that the targetsubstance is released from said ligand to liquid (II).

Between steps (a) and (b) the liquid sample is preferably separated fromthe separatory material which in turn may be washed before step (b).After step (b), liquid (II) may be separated from the separatorymaterial. The target substance, if so desired, may be worked up fromliquid II.

With respect to target substances in form of biological molecules suchas those having nucleotide structure (including nucleic acids),polypeptide structure (including proteins), carbohydrate structure,steroid structure etc the liquids used typically have been aqueous. Thisembodiment of the invention is characterized in that

(i) said separatory material comprises a stimulus-responsive polymer asdefined elsewhere in this specification, which polymer has beenfunctionalized with the ligand, preferably by covalent attachment of theligand after the polymer has been formed, and

(ii) subjecting in step (a) and at least during binding of the targetsubstance to the ligand, the separatory material to a stimulus at alevel/intensity at which the stimulus-responsive polymer in aconformation enhancing binding of the target substance to the ligand,and

(iii) subjecting in step (b) and at least during release of the targetsubstance from the ligand the separatory material to a stimulus at alevel/intensity at which the stimulus-responsive polymer is in aconformation hindering binding of the target substance to the ligand.

The level/intensity of the stimulus is on opposite sides of the criticallevel/intensity for the stimulus-sensitive polymer used and otherconditions applied in the respective step. The process can be madecyclic in case step a is repeated after step b, typically after extrawashing/regeneration steps and equilibration steps.

Various embodiments of the inventive method may be carried out in abatch-wise or a chromatographic mode. Chromatographic modes, forinstance, may be carried out by permitting the various liquids in plugflow (mobile phase) to pass through a bed of the separatory materialwhile subjecting the bed to the appropriate stimulus for the individualsteps and stimulus-responsive polymer used. The bed may be a porousmonolith or a bed of packed or fluidised particles. Batch-wise modes inparticular concerns suspended particles in combination with turbulentflow and/or liquids.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 provides a chromatogram showing the elution of BSA molecules heldon CB molecules from a p(NIPAAm)-AmCB column with temperature change.

FIG. 2 provides a control chromatogram with the use of a p(NIPAAm)column.

DETAILED DESCRIPTION OF INVENTION

The physical stimulus to be used in the method according to the presentinvention is exemplified by temperature. Namely, themolecular-recognizability of a substance interacting with a targetsubstance can be changed under a temperature change by using, forexample, a composite material comprising a heat-responsive polymer. Asan example thereof, a chromatographic packing chemically modified with acomposite material of a polyalkylamide having a terminal functionalgroup, for example, amino, carboxyl or hydroxyl group may be cited. Achemically modified carrier is exemplified by a silica carrier.

Depending on the particular stimulus-responsive polymer used otherstimulus may apply, for instance light, magnetic field, electricalfield, pH, etc. Stimulus-responsive polymers are often called“intelligent polymers”.

Stimulus-responsive polymers are characterized in that they upon beingsubjected to the correct stimulus of the correct intensity or level(critical level of stimulus or critical intensity of stimulus) undergo aconformational and reversible change of their physico-chemicalproperties. The change may be a switch from a pronounced hydrophobicityto a pronounced hydrophilicity or vice versa. The exact level/intensityand kind of the required stimulus depend on the structure of the polymerand will often also depend on other conditions (solvent, solutes such assalts etc). The most well-known and most utilized polymers of this kindrespond to heat (thermo-responsive or temperature-responsive polymers).Temperature-responsive polymers are recognized by having a sharptemperature limit at which they switch from a pronounced hydrophilicstate to a pronounced hydrophobic state and vice versa. Fortemperature-responsive polymer in solution the change inconformation/physico-chemical properties occurs at the so calledcritical solution temperature (CST).

For a temperature responsive polymer in aqueous media there is a lowercritical solution temperature (LCST) or an upper critical solutiontemperature (UCST). For a polymer having a LCST, the polymer change froma hydrophilic conformation to a hydrophobic conformation when thetemperature is passing the LCST from below. For a polymer having anUCST, the change is the opposite when the temperature is passing theUCST from below. The exact value of the LCST and UCST depend on thepolymer and also on other conditions applied (solvent, other solutesetc).

As discussed above one of the characteristic features of the inventionwhen a temperature-sensitive polymer is used is that the binding to andthe release from the ligand is performed at opposite sides of anapplicable CST.

The stimulus-responsive polymer preferably has an insignificant affinityfor the target substance compared to the affinity between the targetsubstance and the covalently attached ligand. Preferably there is nosignificant affinity between the ligand and the thermoresponsivepolymer.

Examples of the fundamental constituent unit of thetemperature-responsive polymer include homopolymers and copolymers ofN-alkyl(meth)acrylamide such as N-isopropyl(meth)acrylamide,N-(meth)acryloylpiperidine, N-propyl(meth)acrylamide,N,N-diethyl(meth)acrylamide, N-cyclopropyl(meth)acrylamide,N-(meth)acryloylpyrrolidine, N,N-ethylmethyl(meth)acrylamide andN-ethyl(meth)acrylamide, and copolymers thereof with monomers containingfunctional groups such as carboxyl, amino, hydroxysuccimido, thiol,imino and epoxy groups for ensuring chemical composition with moleculesinteracting with target substances.

Examples of the polyalkylacrylamide to be used in the method accordingto the present invention include poly(N-isopropylacrylamide)-dyecomposite materials.

Examples of the target substance and the molecule interacting with thetarget substance include biological components composed of amino acids,saccharides, nucleic acids, etc. and organic compounds having amolecular weight of not more than 1,000. Although the amount of theligand molecule to be chemically composed with the stimulus-responsivepolymer may be arbitrarily controlled, it preferably amounts to 0.1 to50% based on the whole composite. The physical of chemical properties ofthe stimulus-responsive polymer can be varied by controlling the amountof the molecule interacting with the target substance to be composedtherewith. For example, a poly(N-isopropylacrylamide) homopolymer has alow limit critical temperature of about 32° C. which can be varied bycontrolling the amount of the molecule interacting with the targetsubstance to be composed therewith.

Separatory Materials (e.g. Chromatographic Packings)

The separatory material to be used in the inventive method comprises abase matrix (carrier) which may be based on organic and/or inorganicmaterial. In case the liquid used is aqueous, the base matrix ispreferably hydrophilic. This in particular applies to target substancesthat are biomolecules of the kind discussed above.

The base matrix is preferably based on a polymer, which preferably isinsoluble and more or less swellable in water. Hydrophobic polymers thathave been derivatized to become hydrophilic are included in thisdefinition. Suitable polymers are polyhydroxy polymers, e.g. based onpolysaccharides, such as agarose, dextran, cellulose, starch, pullulan,etc. and completely synthetic polymers, such as polyacrylic acid amide,polymethacrylic acid amide, poly(hydroxyalkylvinyl ethers),poly(hydroxyalkylacrylates) and polymethacrylates (e.g.polyglycidylmethacrylate), polyvinylalcohols and polymers based onstyrenes and divinylbenzenes, and copolymers in which two or more of themonomers corresponding to the above-mentioned polymers are included.Polymers, which are soluble in water, may be derivatized to becomeinsoluble, e.g. by cross-linking and by coupling to an insoluble bodyvia adsorption or covalent binding. Hydrophilic groups can be introducedon hydrophobic polymers (e.g. on copolymers of monovinyl anddivinylbenzenes) by polymerization of monomers exhibiting groups whichcan be converted to OH, or by hydrophilization of the final polymer,e.g. by adsorption of suitable compounds, such as hydrophilic polymers.

Suitable inorganic materials to be used in base matrices are silica,zirconium oxide, graphite, tantalum oxide etc.

Preferred matrices lack groups that are unstable against hyrolysis, suchas silan, ester, amide groups and groups present in silica as such.

The matrix may be porous or non-porous. This means that the matrix maybe fully or partially permeable (porous) or completely impermeable tothe compound to be removed (non-porous).

The pores may have sizes ≧0.1 μm, such as ≧0.5 μm, by which is meantthat a sphere ≧0.1 μm respective ≧0.5 μm in diameter is able to passthrough. An applied liquid may be able to flow through this kind of poresystem (convective pore system). In case the support matrix is in formof beads packed to a bed, the ratio between the pore sizes of theconvective pore system and the diameter of the particles typically is inthe interval 0.01-0.3, with preference for 0.05-0.2. Pores having sizes≧0.1 μm, such as ≧0.5 μm, are often called macropores.

The base matrix may also have pores with sizes ≦0.5 μm, such as ≦0.1 μmby which is meant that only spheres with diameters ≦0.5 μm, such as ≦0.1μm, can pass through. Pores having sizes ≦0.5 μm, such as ≦0.1 μm, areoften called micropores.

In a particularly interesting embodiment of the present invention, thebase matrix is in the form of irregular or spherical particles withsizes in the range of 1-1000 μm, preferably 5-50 μm for high performanceapplications and 50-300 μm for preparative purposes.

The base matrix may also be in form of a monolith having at leastmacropores as defined above. Alternative geometric forms are theinterior walls of tubes and the like.

The stimulus responsive polymer as defined above may be attached to thebase matrix on its outer surfaces and/or on its interior surfaces(macropore and/or micropore surfaces). It may also be part of thepolymer constituting the base matrix as such. The stimulus responsivepolymer may be attached to the base matrix by physical adsorption and/orcovalent attachment, preferably the latter.

Ligands

Ligands may be attached to the stimulus responsive polymer either beforeor after the polymer has been attached to or incorporated into the basematrix. Attachment to the stimulus polymer may be by affinity bonds orby covalent bonds, preferably the latter. One typical kind of ligandsbinds to the target substance by more or less pure ionic (electrostatic)interactions. Alternatively the binding includes more complexinteractions such as affinity binding (affinity adsorption). For ionicinteractions the ligands comprises positively or negatively chargedentities (ion exchange; the immobilised entity being selected amongprimary, secondary, tertiary and quaternary ammonium, sulphonate,sulphate, phosphonate, phosphate, carboxy etc groups). More complexinteractions are illustrated by the ligand being on of the affinitymembers in the pairs,

(a) antibodies and antigens/haptens,

(b) lectins and carbohydrate structures,

(c) IgG binding proteins and IgG,

(d) polymeric chelators and chelates,

(e) complementary nucleic acids,

Affinity members also include entities participating in catalyticreactions, for instance enzymes, enzyme substrates, cofactors,cosubstrates etc. Members of cell-cell and cell-surface interactions anda synthetic mimetics of bioproduced affinity members are also included.The term ligand also includes more or less complex organic molecules,for instance dyes, that binds through affinity to complex biomolecules,for instance having oligo or polypeptide structure (including proteins),oligo and polynucleotide structure (including nucleic acids), oligo- orpolysaccharide structures etc.

EXAMPLE

The present invention will now be explained in more detail withreference to examples which are not intended to limit the presentinvention.

Example 1

Bovine serum albumin (BSA) used as the target substance and CibacronBlue (CB) as the molecule interacting with the target substance werecomposed with a heat-responsive polymer and the change in interactionwith the target substance by a temperature stimulus was evaluated by achromatographic technique. As a result, it was confirmed that theinteraction changed by a temperature change to dissociate the CBmolecular from BSA.

1. Synthesis of Polymer

(1-1-a) Synthesis of Poly(N-isopropyl Acrylamide/N-acryloxy Succinimide)[Hereinafter Referred to as Poly(IPAAm-co-ASI)] Having Terminal CarboxylGroup.

In a polymerization tube were charged 15 g of N-isopropyl acrylamide,1.24 g of N-acryloxy succinimide as the monomer having a functionalgroup, 0.28 g of mercaptopropionic acid (MPA) as the chain transferagent, 82 mg of 2,2-azobisisobutyronitrile (AIBN) as the polymerizationinitiator and 500 ml of tetrahydrofuran (THF), and the polymerizationtube with the cock closed was placed in liquid nitrogen and completelyfrozen. Then, the cock was opened and the polymerization tube wasdeaerated by a vacuum pump. Subsequently, the cock was closed again andthe polymerization tube containing the reaction solution was placed inpropanol to completely dissolve the sample in the polymerization tube.This operation was repeated three times (freeze-thaw deaeration). Thus,the polymerization tube in which the sample was thoroughly deaerated andwas under reduced pressure was placed in a shaking thermostatic chamberat 70° C. to effect radical polymerization for two hours and as aresult, a copolymer having a carboxyl group at one terminal wasobtained. After the reaction, the copolymer was reprecipitated by addingthe reaction solution dropwise to ice-cooled diethyl ether to obtain apolymer. The resulting polymer was separated by filtration, dried underreduced pressure overnight at normal temperatures, then, dissolved in aTHF solution and purified again in diethyl ether. The polymer thusobtained was separated by filtration, passed through a gel filtrationcolumn to dispense the desired polymer. The resulting polymer wasfreeze-dried, then dissolved in a THF solvent, reprecipitated andseparated by filtration. The polymer thus obtained was dried overnightunder reduced pressure at normal temperature to obtain the desiredpolymer.

(1-1-b) Composing of Aminohexyl Cibacron Blue (Hereinafter Referred toas “AmCB”) With Poly(IPAAm-co-ASI) Having Terminal Carboxyl Group.

In 100 ml of pyridine were dissolved 5.0 g of the synthesized polymerand 0.43 g of aminohexyl Cibacron Blue and stirred at room temperaturefor 24 hours, and 2 ml of isopropylamine was added thereto and theresulting solution was further stirred for 24 hours to composeaminohexyl Cibacron Blue with the polymer. After completion of stirring,the solvent was removed by a rotary evaporator and the compositematerial was freeze-dried. After the drying, the composite material wasdissolved in purified water to dispense the desired composite materialby gel filtration which was then free-dried to obtain the desiredpolymer.

(1-1-c) Active Esterification (Succinylation) of Terminal Carboxyl Groupof Poly(IPAAm-co-ASI) Having Terminal Carboxyl Group/Aminohexyl CibacronBlue(AmCB) Composite Material [Hereinafter Referred to as“p(NIPMm)-AmCB”].

In a 300 ml eggplant-shape flask were placed 3 g of the synthesizedcopolymer, 0.5 g of N,N-dicyclohexylcarbodiimide and 0.5 g ofN-hydroxysuccinimide and dissolved in 100 ml of THH and stirred at roomtemperature for 48 hours. The dicyclohexylurea to be separated out by aby-product during stirring was removed by filtration and the reactionproduct was finally reprecipitated in diethyl ether to obtain ap(NIPAAm)-AmCB composite material in which one terminal wassuccinylated.

(1-1-d) Introduction of Succinylated p(NIPAAm)-AmCB Composite Materialto Carrier (1).

In 100 ml of 1,4-dioxane was dissolved 1.0 g of the succinylatedp(NIPAAm)-AmCB composite material and fixed on aminopropyl silica gel atroom temperature. After stirring the mixture for 24 hours, 1.0 g offresh succinylated p(NIPAAm)-AmCB composite material was dissolved in100 ml of 1,4-dioxane and then reacted with the resulting gel for 24hours. This operation was repeated once again and finally, the reactedgel was separated by filtration and thoroughly washed with an organicsolvent such as dimethylformamide and methanol and a 66.7 mM phosphoricacid buffer solution containing 100 mM to 500 mM NaCl to obtain thedesired packing.

(1-1-e) Introduction of p(NIPAAm)-AmCB Composite Material to Carrier(2).

To 0.65 g of the copolymer whose one terminal was carboxylated wereadded 30 mg of N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone (EEDQ), 0.8g of aminopropyl silica and 100 ml of THF, subjected to nitrogenreplacement for 20 minutes and then, stirred overnight at roomtemperature. After completion of the stirring, silica particles wereseparated by filtration. In the same manner, 0.6 g of the copolymerwhose one terminal was carboxylated, 30 mg ofN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinone (EEDQ) and 100 ml of THFwere added to the resulting silica particles and the mixture wassubjected to nitrogen replacement for 30 minutes and then, stirredovernight at room temperature. After completion of the stirring, silicaparticles were separated by filtration and thoroughly washed with anorganic solvent such as dimethylformamide and methanol and a 66.7 mMphosphoric acid buffer solution to obtain a p(NIPAAm)-AmCB compositematerial-fixed silica column packing.

2. Column Packing

In a packing solvent prepared at a volume ratio ofmethanol:2-propanol:water of 1:1:1 was dispersed 0.6 g of thep(NIPAAm)-AmCB composite material-fixed silica and charged in a columnpacking packer. Packing into a stainless column of 4.6×30 mm was carriedout at a pressure of 100 kg/cm² for the initial 25 ml and at a pressureof 400 kg/cm² for the rest.

3. Preparation of Sample

(3-1) Preparation of Bovine Serum Alubumin (BSA) Sample.

BSA was dissolved in a 66.7 mM phosphoric acid buffer solution having apH of 7.0 to adjust the concentration at 7.60 mg/ml.

4. HPLC Measuring Conditions

Column: p(NIPAAm)-AmCB

Mobile Phase: 66.7 mM phosphoric acid buffer solution having a pH of 7.0

Flow Rate: 0.2 ml/min

Temperature Condition: 40° C. to 2° C.

Measuring Wavelength: 280 nm

5. Results

A typical chromatogram in the case of injecting 20 μl of a 7.6 mg/ml BSAsolution into the p(NIPAAm)-AmCB column is shown in FIG. 1. After 50minutes from the injection, the temperature was lowered from 40° C. to2° C. Further, to equilibrate the temperature at 20° C., feeding of thesolution was stopped for 20 minutes and then, the flow rate was returnedto the initial flow rate of 0.2 ml/min. At 71.25 minutes the elutionpeak was observed and the eluate was brown by micro BCA protein assayand the molecular weight was equal to that of BSA from the results ofthe analysis of the gel filtration chromatography and thus, it wasconfirmed that the elution peak was that of BSC. Further, an experimentof using a silica column to which p(NIPAAm) alone had been fixed wascarried out in the same manner as in using the p(NIPAAm)-AmCB column butBSA did not adhere as and passed through the column shown in FIG. 2. Itwas also confirmed by micro BCA protein assay that the peak at 70.9minutes was not for BSA. Since this peak was also recognized in the caseof a control sample free of BSA, it was thought that this peak was forimpurities. From these results it was confirmed that the interactionbetween a target substance and a molecule interacting with the targetsubstance by a temperature-stimulus could be controlled by a physicalstimulus such as a temperature change.

Industrial Applicability

The separation method with the use of the separatory material accordingto the present invention has the following advantages.

1) Different from the chemical elution methods employed in theconventional chromatographic techniques, no severe chemical condition isneeded therein and thus a useful biopolymer can be recovered at a highyield.

2) By changing an interaction due to a physical stimulus, an interactiondiffering from the inherent one with a target substance can be induced.

3) In the case of the separatory material of the present invention, nopost-elution treatment (desalting, pH regulation, etc.) is needed, as inthe case of the conventional affinity chromatography.

4) A packing can be quickly regenerated, compared with the conventionalaffinity chromatographic carriers.

What is claimed is:
 1. A separation method comprising applying aphysical stimulus to a material containing a ligand interacting with atarget substance, to cause a chemical or physical environmental changein the material wherein the interaction between the ligand and thetarget substance is reversibly changed, thus effecting separation andwherein the material comprises a composite material containing astimulus-responsive polymer and a ligand interacting specifically with atarget substance, wherein said stimulus-responsive polymer undergoes astructural change upon the application of the physical stimulus suchthat the interaction of the target substance and ligand is affected,thereby causing a reversible change in the interaction force with thetarget substance due to the physical stimulus.
 2. The separation methodas claimed in claim 1, wherein said physical stimulus is a temperaturechange.
 3. The separation method as claimed in claim 1, wherein thematerial is a chromatographic packing comprising a carrier having achemically modified surface containing a composite material comprising astimulus-responsive polymer and a ligand interacting specifically with atarget substance.
 4. The separation method as claimed in claim 1,wherein the stimulus-responsive polymer is a polyalkylamide polymer orcopolymer having a terminal functional group.
 5. The separation methodof claim 4, wherein the terminal functional group is an amino group or acarboxyl group.
 6. The method for separating a target substance asclaimed in claim 5, wherein water is used as a mobile phase.
 7. Theseparation method as claimed in claim 1, wherein the physical orchemical properties of the stimulus-responsive polymer are regulated bycontrolling the amounts of the target substance and the ligandinteracting with the target substance in the composite material.